The main objectives of these studies are (1) to identify and characterize all proteins that are specified by the defective human parvovirus (AAV) and to determine similarities and differences with autonomous parvovirus proteins, (2) to define the mechanism(s) by which the AAV proteins arise, and (3) to define specific functions of the AAV proteins. We have identified at least four AAV non-structural proteins. At least one of these proteins is necessary for viral DNA replication. Post-translational processing does not account for production of any AAV structural proteins although they share large segments of sequences-in-common. It is now clear, however, that these proteins originate from independent in-frame initiations. Mechanisms that regulate expression of AAV proteins are of fundamental interest, and we have shown that one AAV structural protein is initiated by a codon (ACG) not known previously to act as an initiation codon in higher eukaryotes. Furthermore, our current findings (i) support a "scanning mechanism" in the translational expression of polycistronic eukaryotic mRNAs, and (ii) demonstrate that alternative mRNA splicing is required for effective translational expression of the largest AAV capsid protein. Among methods used are site-directed mutagenesis, affinity chromatography, gel electrophoresis, in vitro translation of viral RNA, DNA transfection, immunoprecipitation, and Western blotting.